Rapid folding and unfolding of Apaf-1 CARD

Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an alpha-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.     

Role of caspases-3 and -7 in Apaf-1 proteolytic cleavage and degradation events during cisplatin-induced apoptosis in melanoma cells

Apoptosis protease-activating factor-1 (Apaf-1), the central element in the mitochondrial pathway of apoptosis, is frequently absent or poorly expressed in metastatic melanomas, a tumor type showing a low degree of spontaneous apoptosis and a poor response to conventional therapies. In the present study, we used the Apaf-1-positive Me665/2/21 melanoma cell line to investigate the fate of Apaf-1 during cisplatin-induced apoptosis. As novel findings described for the first time in melanoma cells, we observed that Apaf-1 was markedly decreased during apoptosis, already at early stages of cell damage; concurrently, an immunoreactive N-terminal fragment of congruent with 26 kDa was evident. In spite of the remarkable decrease of Apaf-1 in apoptotic cells, caspase-9 was found to be processed and enzymatically active. Both Apaf-1 depletion and its proteolytic cleavage were markedly prevented in presence of the caspase-3/-7 inhibitor ac-DEVD-CHO. In presence of ac-DEVD-CHO, caspase-9 activity was also inhibited, along with a partially different pattern of caspase-9 processing forms. Unexpectedly, the inhibition afforded by ac-DEVD-CHO on several components, that is, caspase-3/-7 and caspase-9 activities, and Apaf-1 proteolytic degradation, did not abrogate the apoptotic morphology and cell detachment, nor the proteolytic degradation of crucial targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. Together, our results suggest that caspase-3 and -7, proved to be dispensable for the above apoptosis-associated events, play a role on Apaf-1 handling and possibly on apoptosome function.     

环境高温促进猪睾丸Apaf-1和Caspase-9的表达

高温热应激条件下,凋亡蛋白表达量升高,生殖细胞凋亡增加。凋亡蛋白酶活化因子1（apoptosis protease activating factor 1,Apaf-1）和凋亡蛋白酶活化起始者含半胱氨酸的天冬氨酸蛋白水解酶9,(cysteine aspartic acid specific protease 9, Caspase-9）是细胞凋亡内源途径中的重要调节蛋白,热应激条件下猪睾丸Apaf-1和Caspase 9的表达未见报道。本研究发现,夏季畜舍高温使Apaf-1和Caspase-9表达量升高。qRT-PCR和Western印迹结果显示,与对照组（正常舍温20℃）相比,短时热应激组（40～42℃,1 h/d, 7 d）和长时热应激组（40～42℃,1 h/d, 42 d）,Apaf-1和Caspase-9 mRNA和蛋白的相对表达量均显著升高。免疫组织化学研究发现,Apaf-1在猪睾丸组织中免疫反应阳性物定位于间质细胞、支持细胞和各个发育阶段生精细胞。热应激处理导致精母细胞和精子细胞Apaf-1表达量升高。在各实验猪睾丸组织中,Caspase-9定位于间质细胞、支持细胞和各个发育阶段生精细胞的胞质中。与对照组相比,热应激处理导致减数分裂以后的生精细胞和支持细胞Caspase-9表达量升高。上述结果表明,高温热应激促进Apaf-1和Caspase-9的表达,提示Apaf-1和Caspase-9表达的变化可能与猪舍高温导致的猪精液品质下降存在关联。     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1

CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA. By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region. Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL. These results indicate that Bcl-XL directly binds to a specific region in Apaf-1.     

Bacillus licheniformis6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌6816中扩增了碱性蛋白酶基因（apr),扩增的1.14kb的DNA片段插入到大肠杆菌载体pET-20b中,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM109（DE3）中得到表达,SDS-PAGE分析显示融合表达产物的分子量为30kD,同核酸序列测定所推导的值相符,表达产物占细胞总蛋白的7.5%,重组菌的酶活比出发菌株提高了3.3倍,研究发现,重组的碱性蛋白酶在进入大肠杆菌周质空间时存在前肽自动脱落的现象。     

人白细胞介素1受体拮抗剂cDNA克隆及其表达影响因素

采用ＰＣＲ技术成功地从人胎盘ｃＤＮＡ文库中扩增出人白细胞介素１受体拮抗剂（ｈＩＬ １ｒａ）成熟蛋白编码区基因,并以此为模板突变其ｃＤＮＡ５;其克隆产物经物理图谱证实与文献报道完全一致,利用含不同ＳＤ的同一表达质粒分别表达含不同５的ｃＤＮＡ,结合文献分析其表达影响因素,同时对ｈＩＬ １ｒａＥ．ｃｏｌｉ表达产物的可能存在形式进行了推论。     

Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli

In this study, a gene coding for thermophilic serine protease of the ClpP class from the thermoacidophilic archaeon Thermoplasma volcanium (Tpv) was cloned and expressed in Escherichia coli. The primary sequence and domain analysis of this enzyme showed similarities (50–60% similarity) to signal peptide peptidases (SppA) of bacteria and other archaea. An increase of about tenfold in the activity was achieved by overexpression of Tpv SppA in E. coli, as detected by enzyme assays conducted using Ala-Ala-Phe-pNa and N-Suc-Ala-Ala-Pro-Phe-pNA as substrates. The recombinant enzyme, purified using an anion exchange column chromatography, displayed an apparent molecular mass of 26 kDa on SDS-PAGE analysis. Purified Tpv SppA was active in a broad range of pH and temperature with maximal activity at 60°C and between pH 7.5 and pH 8.0. The activity of the enzyme was strongly inhibited by inhibitors typical for serine proteases, i.e., chymostatin and PMSF. The activity of the Tpv SppA and the stability at high temperature were significantly enhanced in the presence of 5 mM Ca²⁺ ions. Our multiple sequence alignment data revealed a conserved Ser/Lys catalytic dyad in Tpv SppA that comprised Ser76 (nucleophile) and Lys128 (general base) residues. A search for a transmembrane domain using automated programs did not predict any signal peptide associated with the Tpv SppA and, therefore, suggested a cytoplasmic location for this enzyme.     

胸腺素α_1 基因的克隆表达及其生物活性

 胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 .转化大肠杆菌T0P1 0 ,酶切鉴定正确后 ,经 1mmol LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,用离子交换层析纯化融合蛋白 .溴化氰裂解融合蛋白 ,释放出Tα1单体 ,经离子交换色谱纯化出Tα1.采用3 H TdR参入法进行生物活性测定 ,证实融合蛋白和Tα1均具有刺激小鼠脾淋巴细胞分裂增殖的能力 .     

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.     

海南龙血树WD40转录因子基因DcWD40-1的克隆和表达分析

海南龙血树是国产血竭的主要基源植物,其血竭主要化学成分为类黄酮化合物。为进一步了解DcWD40-1在类黄酮生物合成中的潜在功能和作用机制,该研究根据海南龙血树转录组数据,利用RT-PCR技术在海南龙血树中克隆了一个WD40基因DcWD40-1,该基因全长1 550 bp,包含一个1 353 bp的开放阅读框,编码450个氨基酸,蛋白质分子量50.77 kD,理论等电点5.71。生物信息学分析显示,DcWD40-1属于WD40蛋白家族成员,具有5个保守的WD40结构域,和其他植物WD40蛋白同源性高,保守性强。利用Genome Walking方法分离了1 503 bp的DcWD40-1启动子序列,该区域具有典型真核生物启动子结构特征,并含有多个应答激素和胁迫的响应元件。表达分析显示,血竭诱导剂能够诱导Dc WD40-1的表达,DcWD40-1的变化与血竭形成及类黄酮积累正相关。此外,DcWD40-1也能对茉莉酸甲酯、细胞分裂素、油菜素内酯和UV-B处理做出积极响应。     

胸腺素α_1 基因的克隆表达及其生物活性

胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 .转化大肠杆菌T0P1 0 ,酶切鉴定正确后 ,经 1mmol LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,用离子交换层析纯化融合蛋白 .溴化氰裂解融合蛋白 ,释放出Tα1单体 ,经离子交换色谱纯化出Tα1.采用3 H TdR参入法进行生物活性测定 ,证实融合蛋白和Tα1均具有刺激小鼠脾淋巴细胞分裂增殖的能力 .     

大肠杆菌tktA基因的克隆表达

tktA是芳香族氨基酸生物合成共同途径的关键酶基因之一,在大肠杆菌中,tktA编码转酮酶A,在磷酸戊糖途径生成4－磷酸赤藓糖中起主要作用。采用PCR方法从大肠杆菌K－12株中扩增到tktA,并实现了高效表达,tktA活性提高了3．9倍,并且使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量有所提高。     

地衣芽孢杆菌YP1A耐有机溶剂蛋白酶基因的克隆与功能表达

根据B．licheniformis YP1A来源的碱性蛋白酶具有的高强度耐有机溶剂性能及相关数据库分析,采用PCR克隆B．licheniformis YP1A耐有机溶剂碱性蛋白酶基因,序列分析显示该基因（1264bp）包含启动子与编码380个氨基酸的开放阅读框（ORF）,ORF包括信号肽、前肽及编码254个氨基酸的成熟肽序列。相关基因分析表明,YP1A耐有机溶剂碱性蛋白酶基因与地衣芽孢杆菌ATCC14580的碱性蛋白酶基因仅有6个氨基酸残基差异：构建2种含YP1A碱性蛋白酶CDS的组成型穿梭表达载体pHY／aprYP与pHY／aprP43,前者采用YP1A蛋白酶自带的启动子,后者则采用来自于质粒pP43NMK的P43强启动子。利用这2种表达载体在枯草芽孢杆菌WB800中成功进行蛋白酶的功能表达．其中P43强启动子的表达能力明显优于碱性蛋白酶自带的启动子,表达的蛋白酶比酶活为395U／ml。重组菌表达的碱性蛋白酶在体积分数50％的亲水及疏水有机溶剂中表现出了很好的耐受性,验证了克隆基因为地衣芽孢杆菌YP1A的高强度耐有机溶剂碱性蛋白酶基因.     

MAL1基因启动子区的克隆及其在大肠杆菌中的启动功能分析

目的：MAL1基因启动子区在原核生物大肠杆菌中是否有双向启动外源基因的功能。方法：利用PCR扩增MAL1基因启动子区,将不同方向的MAL1基因启动子区后接上Zeocin基因,构建成重组报告质粒,转化大肠杆菌DH5α,验证该启动子区是否双向都具有启动外源基因的功能。结果：将含不同方向启动子区的重组质粒PUCMZ1和PUCMZ2,转化大肠杆菌,转化子均有Zeoein抗性。结论：证明了MAL1结构基因上游启动子区在原核生物大肠杆菌中具有双向启动的功能,且3′-5′方向的启动能力强于5′-3′方向。     

E.coli木糖异构酶基因的克隆及表达条件的优化

采用PCR技术以大肠杆菌JM109基因组DNA为模板扩增得到木糖异构酶基因xylA,连接到载体pET-22b( ),得到重组质粒pET-22b( )-xylA。将此重组质粒转化到大肠杆菌菌株BL21(DE3)中,重组菌株经IPTG诱导后,通过半胱氨酸-咔唑法测得木糖异构酶活力。每mL发酵液中重组菌株显示出酶活力约为0.84 U。SDS-PAGE电泳结果显示出明显的5×104(相对分子质量)特异性蛋白质条带。